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A molecular ecology approach using Stable Isotope Probing and metagenomics to study viruses of methanogens' diversity Context
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International audience. Viruses of microbes are ubiquitous and they are major drivers of ecosystem functioning [1]. Viral ecology is rapidly developing, driven by the advent of high throughput sequencing [2].Nevertheless, the viral diversity of certain ecosystems is still poorly characterized, such asthose which we are studying in an applied perspective, the anaerobic digestion (AD) plants.AD is an environmental biotechnology which produces methane-rich biogas, a renewable energy source. It relies on the catalytic activity of complex microbial communities. To optimize AD processes, the links between operational parameters, microbial communityproperties and process performance must be finely understood. Indigenous viruses should thus also be considered, as they most certainly influence the matter fluxes during AD. To our knowledge, a unique publication has been dedicated to AD metavirome sequencing [3]; ithighlights their specific diversity compared to metaviromes from other types of ecosystems.Our study focuses on the in situ diversity of DNA viruses of methanogenic archaea, the latter being key players of AD. We are developing an original approach based on stable isotope probing (SIP) [4] and high throughput sequencing. SIP consists in separating DNA molecules according to their density by ultracentrifugation on a CsCl gradient. Using various 13C-labeled methanogenesis substrates enables us to activate selectively distinct populations of methanogens and to enrich them in 13C [5]. The viruses that infects these methanogensshould logically also be labelled with 13C, so that it should be possible to separate their DNA by SIP before sequencing them with a shotgun method.We first proved the concept of SIP application to viral DNA on a pure strain model. We showed by SIP the incorporation of 13C into T4 bacteriophage DNA, obtained by farming onEscherichia coli cells grown in minimal medium, containing either unlabeled or 13C-glucose as sole carbon source. A range of 13C/unlabeled glucose ratios (20% to 80%) was also employed to show that T4 DNA 13C-enrichment is proportional to the 13C glucose rate of the medium.For a first application to AD communities, we selected formate as unique carbon source, expecting to activate Methanobacteriales members as main methanogens. At 3 distinct incubation time points, we prepared cell and virion pellets, extracted their DNA separately, and generated their SIP profiles. For the cellular DNA SIP fractions, we are currently determining the 16S metabarcoding profiles. Viral particles were observed by electron microscopy. In addition to cosmopolitan head-tail virions, morphotypes typical of archaeal viruses were interestingly present, such as spindle shapes (Fuselloviridae, Salterprovirus) [6].[1] Suttle, C.A. Nat. Rev. Microbiol., 5, p.801. (2007)[2] Sullivan, M.B., Weitz, J.S. and Wilhelm, S. Environ. Microbiol. Rep. 9, pp.33-35. (2017)[3] Calusinska, M., Marynowska, M., Goux, X., Lentzen, E., Delfosse, P. Environ. Microbiol. 18, 1162-1175 (2016).[4] Neufeld, J.D., Vohra, J., Dumont, M.G., Lueders, T., Manefield, M., Friedrich, M.W. and Murrell, J.C. Nat. Protoc., 2, p.860 (2007).[5] Li, T., Wu, T.D., Mazéas, L., Toffin, L., Guerquin‐Kern, J.L., Leblon, G. and Bouchez, T. Environ. Microbiol, 10, pp.580-588. (2008)[6] Krupovic, M., Cvirkaite-Krupovic, V., Iranzo, J., Prangishvili, D., Koonin, E. V. Virus Res. 244, 181-193 (2018)
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